Ludomira H. Granicka

ENCAPSULATION OF OKT3 CELLS IN HOLLOW FIBERS

Encapsulation of an OKT3 cell line in hollow fibers was evaluated in vitro and in vivo. The cell line is a mouse hybridoma producing immunoglobulin G2a (IgG2a) against CD3 human T lymphocytes and thus may function as a nonspecific activation system of a subpopulation of human T lymphocytes. For encapsulation purpose, hollow fibers of polypropylene K600 PP Accurel (Akzo, Germany) were selected. Hollow fibers were siliconized to improve membrane biocompatibility for in vivo experiments. The siliconized hollow fibers exhibited acceptable diffusive permeability (P) [ml/min/m2] for small solutes (for creatinine, p = 63.9 +/- 2.0, n = 3) and larger solutes (for albumin, p = 16.9 +/- 1.9, n = 3; for IgG, p = 1.0 +/- 0.2, n = 3). The 12 cm long hollow fibers were filled with a suspension of OKT3 cells of an average density of 10(6) cells/ ml, and both ends were sealed. The encapsulated cells were cultivated in RPMI 1640/10% CS medium at 37 degrees C, 5% CO2 for a period of 3 to 4 days. After the culture period, the medium was tested on human peripheral blood lymphocytes for the presence of anti-CD3 antibody and read in a flow FACS-trac cytometer (Becton Dickinson Immunocytochemistry Systems, San Diego, CA). The tightness of hollow fiber sealing was tested with a bubble point method. The number of cells increased after cultivation by four- to nine-fold on average (n = 11). Ten experiments were performed in vivo with OKT3 cells encapsulated in hollow fibers and implanted subcutaneously into mice for 3 days. In 50% of the experiments, some anti-CD3 antigens on human lymphocytes were found; however, the difference, in comparison with control, in percent of CD3+ was insignificant. In conclusion, the hollow fiber method for cultivation of hybridoma cells in vitro allows for separation of cells from the medium containing secreted anti-CD3 antibodies and is effective in maintaining cell viability. In vivo application needs additional study.

Polypropylene hollow fiber for cells isolation: methods for evaluation of diffusive transport and quality of cells encapsulation.

Formulation of membrane properties is important prior the successful implantation of encapsulated cells producing therapeutically relevant compounds. The purpose of our study was to specify the methods allowing preliminary evaluation of hollow fibers (HF) chosen for immunoisolation. We have selected as estimates (1) diffusive permeability for small and large solutes, and HF cut off (in vitro), (2) histological evaluation of tissue overgrowth after sc. implantation into mice. It was found that diffusive coefficients were linearly dependent on the particle diameter except that of albumin (2–3 times higher than theoretically estimated). This discrepancy imply that for certain particles the interaction with membrane material may be significant. The histological evaluation showed that siliconized HF implanted for 105 days were accepted (there was thin fibrotic layer on the external surface of the HF, no surrounding haemopoietic cells were found). It is concluded that proposed methods for preliminary evaluation of hollow fibers chosen for immunoisolation seems to be reliable and suitable for testing diffusive permeability of each relevant cell product.

The Experimental Study of Polyelectrolyte Coatings Suitability for Encapsulation of Cells

Living cells encapsulated in polymeric shells are receiving increasing attention because of their possible biotechnological and biomedical applications. The aim of this work is to evaluate how different polyelectrolyte coatings, characterized by different numbers of polyelectrolyte layers and by different polyelectrolyte conformations, affect the viability of encapsulated biological material. We demonstrate the ability to individually encapsulate HL-60 cells as well as rat pancreatic islets within polymeric shells consisting of different PE layers using the layer-by-layer process. Coating of HL-60 cells allows for surviving and functioning of cells for all applied PE as well as for different numbers of layers. The islets encapsulated in applied polyelectrolytes exhibited the lower level of mitochondrial activity as compared to non-encapsulated islets. Nevertheless, encapsulated islets exhibited comparable absorbance values during the whole period of culture. Polyelectrolyte coating seems to be a promising way of allowing capsule void volume minimization in a model of encapsulated biological material for local production of biologically active substances.

The Cytotoxic Effect of Polyelectrolyte Shells Coated Bacterial Cells on Human Leukemia Cells

Encapsulation of cells in polymeric shells allowing for separation of biological material from produced factors may find application in the systems for biological processes regulation. Inadequate efficiency of existing therapeutic anticancer regiments and the rise of multi-drug resistant cancer cells have required investigations into novel anticancer strategies. Enhancement of apoptosis in tumors has been suggested as a new anticancer strategy. Pathogenic microorganisms may have the role as the source of agents for apoptotic therapy. Modified cells of Bacillus subtilis were encapsulated using layer-by-layer technique within polymeric shells for application in local anti-tumor therapy. The applied shells were modified with incorporated fullerene derivate to ensure the layers stability and integrity.The impact of modified nano-thin shells coated bacterial cells on human leukemia cells was evaluated in vitro. It was observed that coating with applied polyelectrolyte layers with incorporated fullerenol allowed for bacterial cells functioning during the culture period and the lethal impact on eukaryotic cells was observed. Applied membrane conformation allowing for functioning of encapsulated microorganisms may be recommended or coating shells for local anti-tumor treatment purposes.

Conformal Nano-Thin Modified Polyelectrolyte Coatings for Encapsulation of Cells

Abstract: Encapsulation of cells in polymeric shells allows for separation of biological material from produced factors, which may find biotechnological and biomedical applications. Human T-lymphocyte cell line Jurkat as well as rat pancreatic islets were encapsulated using LbL technique within shells of polyelectrolyte modified by incorporation of biotin complexed with avidin to improve cell coating and to create the potential ability to elicit specific biochemical responses. The coating with nano-thin modified shells allowed for maintenance of the evaluated cells’ integrity and viability during the 8-day culture. The different PE impact may be observed on different biological materials. The islets exhibited lower mitochondrial activity than the Jurkat cells. Nevertheless, coating of cells with polyelectrolyte modified membrane allowed for functioning of both model cell types: 10 μm leukemia cells or 150 μm islets during the culture. Applied membranes maintained the molecular structure during the culture period. The conclusion is that applied modified membrane conformation may be recommended for coating shells for biomedical purposes.

Membrane for immunoisolation-properties before, and post implantation: Preliminary report

The membranes preventing tissue overgrowth as well as toxic influence on cells encapsulated within can be obtained modifying the polypropylene membranes by silanization. The influence of the silanization with different siloxanes on membrane transport properties was assessed before and post implantation. No change in cut-off values was observed. All of the modified membranes delayed tissue overgrowth of implant in mouse. Spectroscopic evaluation of the membrane material after 4, 7 days, 2 and 4 months of implantation revealed membrane material stability. We concluded that evaluated membranes with cells encapsulated within may be applied as the systems for delivery of biologically active substances.

Stabilized nanosystem of nanocarriers with an immobilized biological factor for anti-tumor therapy

Objective The inadequate efficiency of existing therapeutic anti-cancer regiments and the increase in the multidrug resistance of cancer cells underscore the need to investigate novel anticancer strategies. The induction of apoptosis in tumors by cytotoxic agents produced by pathogenic microorganisms is an example of such an approach. Nevertheless, even the most effective drug should be delivered directly to targeted sites to reduce any negative impact on other cells. Accordingly, the stabilized nanosystem (SNS) for active agent delivery to cancer cells was designed for further application in local anti-tumor therapy. A product of genetically modified Escherichia coli, listeriolysin O (LLO), was immobilized within the polyelectrolyte membrane (poly(ethylenimine)|hyaluronic acid) shells of ‘LLO nanocarriers’ coupled with the stabilizing element of natural origin. Methods and results The impact of LLO was evaluated in human leukemia cell lines in vitro. Correspondingly, the influence of the SNS and its elements was assessed in vitro. The viability of targeted cells was evaluated by flow cytometry. Visualization of the system structure was performed using confocal microscopy. The membrane shell applied to the nanocarriers was analyzed using atomic force microscopy and Fourier transform infrared spectroscopy techniques. Furthermore, the presence of a polyelectrolyte layer on the nanocarrier surface and/or in the cell was confirmed by flow cytometry. Finally, the structural integrity of the SNS and the corresponding release of the fluorescent solute listeriolysin were investigated. Conclusion The construction of a stabilized system offers LLO release with a lethal impact on model eukaryotic cells. The applied platform design may be recommended for local anti-tumor treatment purposes.

The use of hollow fiber membranes combined with cytometry in analysis of bacteriological samples.

To avoid destruction of the implanted biological material it may be separated from host immunological system by enclosure within a permiselective membrane. Two-directional diffusion through the membrane of nutrients, metabolic products, as well as bioactive products of encapsulated cells is required to ensure their survival and functional activities. The system of cells encapsulated within the membrane releasing the biologically active substance may be applied either locally to give an opportunity of therapeutic agent activity in the specified place and/or at some convenient site (tissue) for a prolonged period of time.The novel system of bacteria bio-encapsulation using modified membranes, and its assessment by flow cytometry is described and discussed. The encapsulated in membrane bacteria, functioning and releasing their products were evaluated in the systems in vitro and in vivo. The bacteria cells products impact on Eukariotic cells was evaluated. The cytometric evaluation demonstrates the membrane ability to avoid the release of bacteria enclosed within the membrane wall. In experiments with treatment of the bacteria with antibiotic to release products from damaged bacteria it was possible to distinguish stages of the applied antibiotic impact on encapsulated bacteria cells. In E. coli following stages were distinguished: induction of membrane permeability to PI, activation of proteases targeting GFP (protein) and subsequent nucleic acids degradation. In the another experiment the evidence was presented of the cytotoxic activity of live Bacillus subtilis encapsulated within the membrane system. The Bacilus products mediated by secreted listeriolysin O (LLO) on the chosen eukaryotic cells was evaluated. Similar systems releasing bacterial products locally and continuously may selectively affect different types of cells and may have possible application in the anticancer treatment at localized sites.

Gold Nanoparticle-Modified Poly(vinyl chloride) Surface with Improved Antimicrobial Properties for Medical Devices

Despite the significant technological progress achieved in the past decades in the medical field, device-related infections carry a heavy social and economic burden. Surface modification of medical equipment is one of the most interesting approaches employed to improve the antibacterial activity of a material. Herein, we developed a process for the gold nanoparticle modification of a poly(vinyl chloride) laryngeal tube, which typically serves as an airway management device. In our study, we focused specifically on increasing the antimicrobial properties of the material while maintaining its biocompatibility. We applied two different modification methods to the poly(vinyl chloride) laryngeal tube. An increase in the antimicrobial activity of the surface was observed for both methods. In addition, the adsorption of bacterial cells on the material surface was assessed. We determined that the number of colonies cultured in the presence of the gold nanoparticle-modified samples or absorbed to the material surface decreased significantly compared with the control group. The trend was observed for both Gram-positive and Gram-negative strains. Moreover, it was established that the designed material did not exhibit a lethal impact on a control cell line. Finally, we noted discrepancies in the growth of bacteria cultured in the presence of modified or unmodified PVC material as well as differences in cell adherence to its surface. The proposed poly(vinyl chloride) modifications are most effective against Gram-positive bacteria, especially L. monocytogenes. Nevertheless, it ought to be emphasized that due to their different properties, each strain requires an individual approach.

Cryopreservation of Cells Encapsulated Within Nano-thin Polyelecrolyte Coatings

Cryopreservation is a method which enables to store the cells for a long time period and allows to obtain the appropriate amount of cells necessary for transplantation. Unfortunately, the cells isolated from organs like e.g. hepatocytes are susceptible to freezing damage. Encapsulation may be considered as a method allowing to protect cells during adverse freezing conditions.